采用哺乳动物细胞表面展示技术构建全长人源膀胱癌特异性抗体基因库
首发时间:2013-03-15
摘要:目的 构建大容量且能高效展示于哺乳动物细胞表面的全长人源膀胱癌特异性抗体基因库。方法:分离膀胱癌患者的外周血单核细胞(PBMC),提取PBMC的总RNA,用RT-PCR方法扩增全套IgG1重链可变区(VH)和Kappa 型轻链全长(LCκ)基因,经过相应的限制性内切酶酶切后分别插入pDGB-HC-TM载体,分别构建膀胱癌特异性的抗体重链基因库和轻链基因库(一级抗体库)。随机挑取20个单克隆测定抗体基因序列,并瞬时转染FCHO细胞,结合流式细胞仪检测细胞表面抗体的表达。将上述构建好的轻重链基因库通过4片段连接方法插入双表达载体pDGB4,构建二级抗体库,转染FCHO后用流式细胞仪检测细胞表面抗体表达。结果:分别成功构建了膀胱癌特异性的重链基因库和轻链基因库,随机挑取的轻重链单克隆各有7个和9个含有正确的抗体基因编码序列,且能展示于哺乳动物细胞表面,理论库容量达到3.32×10^11[(1.7×10^6×70%)×(3.1×10^5×90%)]。成功构建了膀胱癌二级抗体库,库容量为9×10^5。结论:利用哺乳动物细胞表面展示技术,我们成功构建了膀胱癌特异性的全长人源抗体基因库,一级抗体库的库容量达到3.32×10^11,二级抗体库库容量达到9×10^5,为下一步筛选特异性、高亲和力抗膀胱癌抗体奠定了基础。
关键词: 膀胱癌 哺乳动物细胞表面展示 全长人源抗体 流式细胞仪检测
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Construction of full-length human bladder cancer-specific antibody libraries based on mammalian display technology
Abstract:Objective: To construct full-length human bladder cancer-specific antibody libraries which display full-length antibodies on the surfaces of mammalian cells. Methods: Total RNA was isolated from peripheral blood mononuclear cells which were from patients with bladder cancer; The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific; The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chain and light chain; 10 clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells for analysis of antibody display on mammalian surfaces by flow cytometry after stained with corresponding fluorescent labeled antibodies. Results: The libraries of bladder cancer specific antibody heavy chain(IgG1) and light chain (LCk) were successfully constructed, 7 out of 10 clones randomly picked up from HC library and 9 out of 10 clones from LC library have correct reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reaches 3.32×10^11 [(1.7×10^6×70%) ×(3.1×10^5×90%)] .Conclusion: We successfully constructed a full-length human bladder cancer specific antibody library with a combinatory diversity of 3.32×10^11 based on mammalian display technology, which can be used for the screening of monoclonal antibody against bladder-cancer-associated antigens.
Keywords: Bladder cancer Mammalian cell display Full-length human antibody Flow cytometry
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